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International audience; Culex pipiens mosquitoes are infected with Wolbachia (wPip) that cause an important diversity of cytoplasmic incompatibilities (CIs). Functional transgenic studies have implicated the cidA-cidB operon from wPip and its homolog in wMel in CI between infected Drosophila males and uninfected females. However, the genetic basis of the CI diversity induced by different Wolbachia strains was unknown. We show here that the remarkable diversity of CI in the C. pipiens complex is due to the presence, in all tested wPip genomes, of several copies of the cidA-cidB operon, which undergoes diversification through recombination events. In 183 isofemale lines of C. pipiens collected worldwide, specific variations of the cidA-cidB gene repertoires are found to match crossing types. The diversification of cidA-cidB is consistent with the hypothesis of a toxin–antitoxin system in which the gene cidB co-diversifies with the gene cidA, particularly in putative domains of reciprocal interactions.

Bacteria of the Bacillus genus are able to synthesize several families of lipopeptides. These small molecules are the product of non-ribosomal peptide synthetases. In 2000, it was found that Bacillus thuringiensis, an entomopathogenic bacterium of the Bacillus cereus group, produced a previously unknown lipopeptide: kurstakin. Genomic analyses reveal that the krs locus, encoding the kurstakin synthetases, is specific to the B. cereus group, but is unevenly distributed within this group. Previous work showed that krs transcription requires the necrotrophism quorum-sensor NprR. Here, we demonstrated that the genes of the krs locus form an operon and we defined its transcription start site. Following krs transcription at the population and single-cell levels in multiple culture conditions, we depicted a condition-dependent transcription pattern, indicating that production of kurstakin is subject to environmental regulation. Consistent with this idea, we found krs transcription to be regulated by another master regulator, Spo0A, suggesting that krs expression is fine-tuned by integrating multiple signals. We also reported an unknown DNA palindrome in the krs promoter region that modulates krs expression. Due to their surfactant properties, lipopeptides could play several physiological roles. We showed that the krs locus was required for proper biofilm structuration.

International audience; The epidemiology of meningococcal disease varies by geography and time. Whole-genome sequencing of Neisseria meningitidis serogroup X isolates from sub-Saharan Africa and Europe showed that serogroup X emergence in sub-Saharan Africa resulted from expansion of particular variants within clonal complex 181. Virulence of these isolates in experimental mouse models was high.

International audience; The bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure.

The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC . This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

The carbon storage regulator protein CsrA regulates cellular processes post-transcriptionally by binding to target-RNAs altering translation efficiency and/or their stability. Here we identified and analyzed the direct targets of CsrA in the human pathogen Legionella pneumophila. Genome wide transcriptome, proteome and RNA co-immunoprecipitation followed by deep sequencing of a wild type and a csrA mutant strain identified 479 RNAs with potential CsrA interaction sites located in the untranslated and/or coding regions of mRNAs or of known non-coding sRNAs. Further analyses revealed that CsrA exhibits a dual regulatory role in virulence as it affects the expression of the regulators FleQ, LqsR, LetE and RpoS but it also directly regulates the timely expression of over 40 Dot/Icm substrates. CsrA controls its own expression and the stringent response through a regulatory feedback loop as evidenced by its binding to RelA-mRNA and links it to quorum sensing and motility. CsrA is a central player in the carbon, amino acid, fatty acid metabolism and energy transfer and directly affects the biosynthesis of cofactors, vitamins and secondary metabolites. We describe the first L. pneumophila riboswitch, a thiamine pyrophosphate riboswitch whose regulatory impact is fine-tuned by CsrA, and identified a unique regulatory mode of CsrA, the active stabilization of RNA anti-terminator conformations inside a coding sequence preventing Rho-dependent termination of the gap operon through transcriptional polarity effects. This allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Thus the L. pneumophila genome has evolved to acclimate at least five different modes of regulation by CsrA giving it a truly unique position in its life cycle., Author summary The RNA binding protein CsrA is the master regulator of the bi-phasic life cycle of Legionella pneumophila governing virulence expression in this intracellular pathogen. Here, we have used deep sequencing of RNA enriched by co-immunoprecipitation with epitope-tagged CsrA to identify CsrA-associated transcripts at the genome level. We found 479 mRNAs or non-coding RNAs to be targets of CsrA. Among those major regulators including FleQ, the regulator of flagella expression, LqsR, the regulator of quorum sensing and RpoS implicated in stress response were identified. The expression of over 40 type IV secreted effector proteins important for intracellular survival and virulence are under the control of CsrA. Combined with transcriptomics, whole shotgun proteomics of a wild type and a CsrA mutant strain and functional analyses of several CsrA-targeted RNAs we identified the first riboswitch in L. pneumophila, a thiamine pyrophosphate riboswitch, and discovered a new mode of regulation by CsrA that allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Our results further underline the indispensable role of CsrA in the life cycle of L. pneumophila and provide new insights into its regulatory roles and mechanisms.

International audience; The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3-to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfpas reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5′ promoter region is required for maximal transcription of gfpas a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110AcovR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

International audience; Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.

International audience; We identified the genes encoding the proteins for the transport of glucose and maltose in Neisseria meningitidis strain 2C4-3. A mutant deleted for NMV_1892(glcP) no longer grew on glucose and deletion of NMV_0424(malY) prevented the utilization of maltose. We also purified and characterized glucokinase and α-phosphoglucomutase, which catalyze early catabolic steps of the two carbohydrates. N. meningitidis catabolizes the two carbohydrates either via the Entner-Doudoroff (ED) pathway or the pentose phosphate pathway, thereby forming glyceraldehyde-3-P and either pyruvate or fructose-6-P, respectively. We purified and characterized several key enzymes of the two pathways. The genes required for the transformation of glucose into gluconate-6-P and its further catabolism via the ED pathway are organized in two adjacent operons. N. meningitidis also contains genes encoding proteins which exhibit similarity to the gluconate transporter (NMV_2230) and gluconate kinase (NMV_2231) of Enterobacteriaceae and Firmicutes. However, gluconate might not be the real substrate of NMV_2230 because N. meningitidis was not able to grow on gluconate as the sole carbon source. Surprisingly, deletion of NMV_2230 stimulated growth in minimal medium in the presence and absence of glucose and drastically slowed the clearance of N. meningitidis cells from transgenic mice after intraperitoneal challenge.

International audience; Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.

International audience; The bacterium Legionella pneumophila is found ubiquitously in aquatic environments and can cause a severe pneumonia in humans called Legionnaires' disease. How this bacterium switches from intracellular to extracellular life and adapts to different hosts and environmental conditions is only partly understood. Here we used RNA deep sequencing from exponentially (replicative) and post exponentially (virulent) grown L. pneumophila to analyze the transcriptional landscape of its entire genome. We established the complete operon map and defined 2561 primary transcriptional start sites (TSS). Interestingly, 187 of the 1805 TSS of protein-coding genes contained tandem promoters of which 93 show alternative usage dependent on the growth phase. Similarly, over 60% of 713 here identified ncRNAs are phase dependently regulated. Analysis of their conservation among the seven L. pneumophila genomes sequenced revealed many strain specific differences suggesting that L. pneumophila contains a highly dynamic pool of ncRNAs. Analysis of six ncRNAs exhibiting the same expression pattern as virulence genes showed that two, Lppnc0584 and Lppnc0405 are indeed involved in intracellular growth of L. pneumophila in A. castellanii. Furthermore, L. pneumophila encodes a small RNA named RsmX that functions together with RsmY and RsmZ in the LetA-CsrA regulatory pathway, crucial for the switch to the virulent phenotype. Together our data provide new insight into the transcriptional organization of the L. pneumophila genome, identified many new ncRNAs and will provide a framework for the understanding of virulence and adaptation properties of L. pneumophila.

Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis., Author summary In Bacillus subtilis, sporulation, competence and biofilm formation are regulated by a pleiotropic regulator called SinR. Two sinR homologs are present in C. difficile genome as an operon and henceforth labeled as sinR and sinR’. Our detailed investigation revealed that in C. difficile, the SinR and SinR’ are key master regulators needed for the regulation of several pathways including sporulation, toxin production, and motility.

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes.

International audience; Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contribute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes.

Shigella , the leading cause of bacillary dysentery, uses a type III secretion system (TTSS) to inject proteins into human cells, leading to bacterial invasion and a vigorous inflammatory response. The bacterium is protected against the response by the O antigen of lipopolysaccharide (LPS) on its surface. We show that bacteriophage-encoded glucosylation of Shigella O antigen, the basis of different serotypes, shortens the LPS molecule by around half. This enhances TTSS function without compromising the protective properties of the LPS. Thus, LPS glucosylation promotes bacterial invasion and evasion of innate immunity, which may have contributed to the emergence of serotype diversity in Shigella .

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti , a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu , for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes ( ehuA , ehuB , ehuC , and ehuD ) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB , as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

Journal Article; Review; Group B streptococcus [(GBS or Streptococcus agalactiae)] is a leading cause of neonatal meningitis and septicaemia. Most clinical isolates express simultaneously a β-haemolysin/cytolysin and a red polyenic pigment, two phenotypic traits important for GBS identification in medical microbiology. The genetic determinants encoding the GBS haemolysin and pigment have been elucidated and the molecular structure of the pigment has been determined. The cyl operon involved in haemolysin and pigment production is regulated by the major two-component system CovS/R, which coordinates the expression of multiple virulence factors of GBS. Genetic analyses indicated strongly that the haemolysin activity was due to a cytolytic toxin encoded by cylE. However, the biochemical nature of the GBS haemolysin has remained elusive for almost a century because of its instability during purification procedures. Recently, it has been suggested that the haemolytic and cytolytic activity of GBS is due to the ornithine rhamnopolyenic pigment and not to the CylE protein. Here we review and summarize our current knowledge of the genetics, regulation and biochemistry of these twin GBS phenotypic traits, including their functions as GBS virulence factors. Yes

International audience; Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.

International audience; Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways , some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO-haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mecha-nistic model for its dual regulatory role.

International audience; The emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen.

International audience; Legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two-hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. Indeed, mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not circadian KaiB1C1. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

International audience; The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N- and C-terminal halves of the ATP-dependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI- and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-specific transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.

International audience; The clpP genes are widespread among living organisms and encode the proteolytic subunit of the Clp ATP-dependent protease. These genes are present in a single copy in most eubacteria. However, five clpP genes were identified in Streptomyces coelicolor. The clpP1 clpP2 operon was studied: mutations affected the growth cycle in various Streptomyces. Here, we report studies of the expression of the clpP3 clpP4 operon in Streptomyces lividans. The clpP3 operon was induced in a clpP1 mutant strain, and the regulation of expression was investigated in detail. The product of the putative regulator gene, downstream from clpP4, was purified. Gel migration shift assays and DNase I footprinting showed that this protein binds to the clpP3 promoter and recognizes a tandem 6 bp palindromic repeat (TCTGCC-3N-GGCAGA). In vivo, this DNA-binding protein, named PopR, acts as an activator of the clpP3 operon. Studies of popR expression indicate that the regulator is probably controlled at the post-transcriptional level.

International audience; Dickeya dadantii is a plant pathogen that secretes cell wall-degrading enzymes (CWDE) that are responsible for soft-rot symptoms. Virulence genes are expressed in a concerted manner and culminate when bacterial multiplication slows. We identify a 25 kb vfm cluster required for D. dadantii CWDE production and pathogenesis. The vfm cluster encodes proteins displaying similarities both with enzymes involved in amino acid activation and with enzymes involved in fatty acid biosynthesis. These similarities suggest that the vfm genes direct the production of a metabolite. Cell-free supernatant from the D. dadantii wild-type strain restores CWDE production in vfm mutants. Collectively, our results indicate that vfm genes direct the synthesis of an extracellular signal and constitute a new quorum sensing system. Perception of the signal is achieved by the two-component system VfmH-VfmI, which activates the expression of the vfmE gene encoding an AraC regulator. VfmE then activates both the transcription of the CWDE genes and the expression of the vfm operons. The vfm gene cluster does not seem to be widespread among bacterial species but is conserved in other Dickeya species and could have been laterally transferred to Rahnella. This work highlights that entirely new families of bacterial languages remain to be discovered.

International audience; The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein. FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3'. In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family. On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator. However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR.

International audience; For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH

International audience; Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface-associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle-biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase-regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid-associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co-ordinate basic metabolism, virulence and modifications of lifestyle.

International audience; ObjectivesTo determine the complete nucleotide sequence of the VIM-1-encoding plasmid pTC2, which was isolated from a Greek Providencia stuartii multiresistant strain.MethodsThe pTC2 plasmid was extracted and sequenced using shotgun and 3 kb paired-end DNA libraries and a 454 sequencing approach. Following assembly into a unique scaffold, gaps were closed by PCR followed by Sanger DNA sequencing. Gene predictions and annotations were performed using the CAAT-Box tool and the Conserved Domain Search service. Sequence comparisons were performed using the Artemis Comparison Tool.ResultsPlasmid pTC2 (180 184 bp) was found to be a multireplicon plasmid (IncA/C and IncR), with a large IncA/C backbone and a mosaic multidrug resistance (MDR) region, in which was inserted a 13 kb IncR fragment. Gene annotation allowed the identification of a complete IncA/C-type transfer system and of several putative maintenance modules, both on the IncA/C backbone and on the IncR fragment. The complex MDR region contained 9 insertion sequences (7 IS26, 1 IS1 and 1 IS6100), 10 resistance genes and a mercury resistance operon integrated into unit transposons, composite transposons or integrons.ConclusionsThe pTC2 combines a broad host range, transfer and maintenance capacities, plasticity of the MDR region and a wide variety of resistance genes, properties that may contribute to the spreading of resistance determinants.

International audience; The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

International audience; The ability of Neisseria meningitidis to establish efficient interaction with host cells is crucial for its survival. We recently demonstrated that an entire operon containing genes NMA1802 to NMA1806 was overexpressed during the early stage of the colonization process. In this work, we investigated whether upregulation of the expression of this operon facilitated the ability of N. meningitidis to adapt to growth on host cells. Using a strain displaying an inducible operon, we demonstrated that the NMA1802-NMA1806 cell-contact-regulated operon could potentially improve the adaptability of meningococcus during growth on the cell surface through enhanced generation of variants.

International audience; BACKGROUND: Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. METHODS: S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. RESULTS: We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. CONCLUSIONS: This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

International audience; Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

The stationary phase sigma factor σ(S) (RpoS) controls a regulon required for general stress resistance of the closely related enterobacteria Salmonella and Escherichia coli. The σ(S)-dependent yncC gene encodes a putative DNA binding regulatory protein. Application of the surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) ProteinChip technology for proteome profiling of wild-type and mutant strains of Salmonella enterica serovar Typhimurium revealed potential protein targets for YncC regulation, which were identified by mass spectrometry, and subsequently validated. These proteins are encoded by the σ(S)-dependent operon yciGFEkatN and regulation of their expression by YncC operates at the transcriptional level, as demonstrated by gene fusion analyses and by in vitro transcription and DNase I footprinting experiments with purified YncC. The yciGFE genes are present (without katN) in E. coli K-12 but are poorly expressed, compared with the situation in Salmonella. We report that the yciGFE(katN) locus is silenced by the histone-like protein H-NS in both species, but that σ(S) efficiently relieves silencing in Salmonella but not in E. coli K-12. In Salmonella, YncC acts in concert with σ(S) to activate transcription at the yciG promoter (pyciG). When overproduced, YncC also activated σ(S)-dependent transcription at pyciG in E. coli K-12, but solely by countering the negative effect of H-NS. Our results indicate that differences between Salmonella and E. coli K-12, in the architecture of cis-acting regulatory sequences upstream of pyciG, contribute to the differential regulation of the yciGFE(katN) genes by H-NS and YncC in these two enterobacteria. In E. coli, this locus is subject to gene rearrangements and also likely to horizontal gene transfer, consistent with its repression by the xenogeneic silencer H-NS.

Between 1942 and 1956, Jacques Monod, Nobel Prize winner in Physiology/Medicine, contributed a number of papers to the Annales de l' Institut Pasteur, the ancestor of the journal "Research in Microbiology". Circumstances that led him to publish in the "Annales" are recalled here. Entre 1942 et 1956, Jacques Monod, Prix Nobel de physiologie et de médecine a rédigé de nombreux articles dans les Annales de l'Institut Pasteur, ancêtre du journal "Research in Microbiology". Les circonstances qui l'ont amené à publier dans les "Annales" sont rappelées ici.

Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54) of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism.

Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus . Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Δ stk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.

In Streptococcus pneumoniae , stkP and phpP , encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called “soluble” protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone “competence stimulating peptide.” We show that PhpP activity is essential in a stkP + genetic background, suggesting tight control of StkP activity by PhpP. Proteins PhpP and StkP colocalized to the cell membrane subcellular fraction and likely belong to the same complex, as revealed by coimmunoprecipitation in cellular extracts. Specific coimmunoprecipitation of the N-kinase domain of StkP and PhpP recombinant proteins by PhpP-specific antibodies demonstrates direct interaction between these proteins. Consistently, flow cytometry analysis allowed the determination of the cytoplasmic localization of PhpP and of the N-terminal kinase domain of StkP, in contrast to the periplasmic localization of the StkP C-terminal PASTA ( p enicillin-binding protein a nd s erine- t hreonine kinase a ssociated) domain. A signaling route involving interplay between serine, threonine, and histidine phosphorylation is thus described for the first time in this human pathogen.

International audience; Staphylococcus aureus necrotizing pneumonia is a severe disease caused by S. aureus strains carrying the Panton Valentine leukocidin (PVL) genes (lukS-PV & lukF-PV) encoded on various bacteriophages (such as phiSLT). Clinical PVL+ strains isolated from necrotizing pneumonia display an increased attachment to matrix molecules (type I and IV collagens and laminin), a phenotype that could play a role in bacterial adhesion to damaged airway epithelium during the early stages of necrotizing pneumonia (J Infect Dis 2004; 190: 1506-15). To investigate the basis of the observed adhesion of S. aureus PVL+ strains, we compared the ability of PVL+ and their isogenic PVL(-) strains to attach to various immobilized matrix molecules. The expression of recombinant fragments of the PVL subunits and the addition of synthetic peptides indicated that the processed LukS-PV signal peptide (LukS-PV SP) was sufficient to significantly enhance the ability of S. aureus to attach to extracellular matrix (ECM) components. Furthermore, we showed that adhesion to ECM components was inhibited by heparin and heparan sulfates (HS) suggesting that in vivo, HS could function as a molecular bridge between the matrix and S. aureus expressing the LukS-PV signal peptide. Site directed mutagenesis, biochemical and structural analyses of the LukS-PV signal peptide indicate that this peptide is present at the S. aureus surface, binds to HS in solid phase assay, and mediates the enhanced S. aureus matrix component adhesion. Our data suggests that after its cleavage by signal peptidase, the signal peptide is released from the membrane and associates to the cell wall through its unique C-terminus sequence, while its highly positively charged N-terminus is exposed on the bacterial surface, allowing its interaction with extracellular matrix-associated HS. This mechanism may provide a molecular bridge that enhances the attachment of the S. aureus PVL+ strains to ECM components exposed at damaged epithelial sites.

International audience; In order to survive, bacteria have developed avariety of highly sophisticated and sensitive signal transduction pathways with which they adapt their genetic expression to meet the challenges of their ever-changing surroundings. These mechanisms enable bacterial cells to communicate with their environment, their hosts and each other, allowing them adopt specific responses, or develop specialised structures such as biofilms or spores to ensure survival, colonization of their ecological niches and dissemination. As highlighted in this book, the so-called two-component systems (TCSs) are one of the most widespread and efficient strategies used for this purpose, where signal acquisition involves autophosphorylation of a sensor histidine kinase and transduction takes place when the kinase phosphorylates its cognate response regulator protein, leading in turn to specific alteration ofgene expression. In their simplest form, TCSs elegantly combine sensing, transducing and transcription activation modules within two proteins, effectively coupling external signals to genetic adaptation. The high degree of conservation among TCS phosphotransfer domains, their ubiquitous nature and the fact that several are essential for cell viability has made them an attractive target for novel classes of antimicrobial compounds. The WalK/WalR (aka YycG/YycF) two-component system, originally identified in Bacillus subtilis, is very highly conserved and specific to low G + C Gram-positive bacteria, including several pathogens such as Staphylococcus aureus. While this system is essential for cell viability, both the nature of its regulon and its physiological role had remained mostly uncharacterized. A number of recent studies have now unveiled a conserved function for this system in different bacteria, defining this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR. This review will focus on the cellular function of the WalK/WalR TCS in different bacterial species and the attractive target it constitutes for novel classes of antimicrobial compounds.

Background Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain χ7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. Results Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain χ7122. Conclusion Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence.

International audience; The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR). The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR. By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator. DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes. We show that the degS and degU genes are organized in an operon. The putative sigma A promoter of the operon was mapped upstream from degS. Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied. The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation. These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.

Transport of maltose in Lactobacillus casei BL23 is subject to regulation by inducer exclusion. The presence of glucose or other rapidly metabolized carbon sources blocks maltose transport by a control mechanism that depends on the phosphorylation of the HPr protein at serine residue 46. We have identified the L. casei gene cluster for maltose/maltodextrin utilization by sequence analysis and mutagenesis. It is composed of genes coding for a transcriptional regulator, oligosaccharide hydrolytic enzymes, an ABC transporter (MalEFGK2) and the enzymes for the metabolism of maltose or the degradation products of maltodextrins: maltose phosphorylase and beta-phospho-glucomutase. These genes are induced by maltose and repressed by the presence of glucose via the catabolite control protein A (CcpA). A mutant strain was constructed which expressed the hprKV267F allele and therefore formed large amounts of P-Ser-HPr even in the absence of a repressive carbon source. In this mutant, transport of maltose was severely impaired, whereas transport of sugars not subject to inducer exclusion was not changed. These results strengthen the idea that P-Ser-HPr controls inducer exclusion and make the maltose system of L. casei a suitable model for studying this process in Firmicutes.

Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development. Escherichia coli is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of E. coli by taking control of two biofilm-promoting factors, autotransported antigen 43 adhesin and the bscABZC cellulose operon. The modulation of the biofilm formation capacities of E. coli or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia. © 2007 Elsevier Ltd All rights reserved.

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc 2 155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.

The uracil salvage pathway in Lactobacillus plantarum was demonstrated to be dependent on the upp-pyrP gene cluster. PyrP was the only high-affinity uracil transporter since a pyrP mutant no longer incorporated low concentrations of radioactively labeled uracil and had increased resistance to the toxic uracil analogue 5-fluorouracil. The upp gene encoded a uracil phosphoribosyltransferase (UPRT) enzyme catalyzing the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate to UMP and pyrophosphate. Analysis of mutants revealed that UPRT is a major cell supplier of UMP synthesized from uracil provided by preformed nucleic acid degradation. In a mutant selection study, seven independent upp mutants were isolated and all were found to excrete low amounts of pyrimidines to the growth medium. Pyrimidine-dependent transcription regulation of the biosynthetic pyrimidine pyrR1-B-C-Aa1-Ab1-D-F-E operon was impaired in the upp mutants. Despite the fact that upp and pyrP are positioned next to each other on the chromosome, they are not cotranscribed. Whereas pyrP is expressed as a monocistronic message, the upp gene is part of the lp _ 2376-glyA-upp operon. The lp _ 2376 gene encodes a putative protein that belongs to the conserved protein family of translation modulators such as Sua5, YciO, and YrdC. The glyA gene encodes a putative hydroxymethyltransferase involved in C 1 unit charging of tetrahydrofolate, which is required in the biosynthesis of thymidylate, pantothenate, and purines. Unlike upp transcription, pyrP transcription is regulated by exogenous pyrimidine availability, most likely by the same mechanism of transcription attenuation as that of the pyr operon.

The concentration of GABA increases rapidly in wounded plant tissues, but the implication of this GABA pulse for plant–bacteria interactions is not known. Here we reveal that GABA stimulated the inactivation of the N -(3-oxooctanoyl)homoserine lactone (OC8-HSL) quorum-sensing signal (or “quormone”) by the Agrobacterium lactonase AttM. GABA induced the expression of the attKLM operon, which was correlated to a decrease in OC8-HSL concentration in Agrobacterium tumefaciens cultures. The Agrobacterium GABA transporter Bra was required for this GABA-signaling pathway. Furthermore, transgenic tobacco plants with elevated GABA levels were less sensitive to A. tumefaciens C58 infection than were wild-type plants. These findings indicate that plant GABA may modulate quorum sensing in A. tumefaciens , thereby affecting its virulence on plants. Whereas GABA is an essential cell-to-cell signal in eukaryotes, here we provide evidence of GABA acting as a signal between eukaryotes and pathogenic bacteria. The GABA signal represents a potential target for the development of a strategy to control the virulence of bacterial pathogens.

It has been shown previously that expression of theStreptomyces lividans clpP1P2operon, encoding proteolytic subunits of the Clp complex, theclpC1gene, encoding the ATPase subunit, and thelongene, encoding another ATP-dependent protease, are all activated by ClgR. The ClgR regulon also includes theclgRgene itself. It is shown here that the degradation of ClgR and Lon is ClpP1/P2-dependent and that the two C-terminal alanines of these new substrates are involved in their stability. The ClpC1 protein, which does not end with two alanines, is also accumulated in aclpP1P2mutant. The results presented here support the idea that ClpP1/P2 ensure post-translational control of ClgR regulon members, including ClgR itself.